The present invention relates to a method for quantitative measurement of a substrate such as sucrose, glucose, etc. contained in a sample based on enzyme reaction, and to an analysis element used therefor.
Currently, there is an increasing demand for a method that facilitates simplified quantitative analysis of blood sugar and urine sugar levels at home.
Conventionally developed methods for quantitating sugars such as sucrose, glucose, etc. use polarimetry, colorimetry, reduction titration and a variety of chromatography. All the prior methods, however, have a drawback that their application at home with ease is difficult. For example, the method using polarimetry permits simplified manipulation but is greatly affected by the temperature at measurement. This method has another drawback of being difficult to make accurate measurement of blood polarization with a simple instrument. Moreover, all the above-mentioned conventional methods have only poor accuracy due to their lower specificity to sugars.
Under the circumstance, a biosensor that utilizes specific catalytic action of enzyme has been developed in order to facilitate simplified quantitation of sugar level at home.
In the following, a method for quantitating glucose will be described as one example of the method for quantitative measurement of sugar level using a biosensor.
A commonly known method for electrochemically quantitating glucose is a method which combines glucose oxidase (EC1.1.3.4; hereinafter abbreviated to "GOD") as an enzyme with an oxygen electrode or hydrogen peroxide electrode (for example, "Biosensor", edit by Shuichi Suzuki, published by Kodansha).
GOD can selectively oxidize a substrate .beta.-D-glucose to D-glucono-.delta.-lactone in the presence of an electron acceptor. If present, oxygen can function as an electron acceptor and is reduced to hydrogen peroxide by GOD-mediated oxidation reaction. This means that the decreased amount of oxygen or increased amount of hydrogen peroxide is proportional to the content of glucose in a sample. Therefore, glucose concentration in a sample can be quantitated by reacting a glucose containing sample with an enzyme in the presence of oxygen and measuring the resultant decrease in oxygen amount using the oxygen electrode or the resultant increase in hydrogen peroxide amount using the hydrogen peroxide electrode.
However, as speculated from the reaction of the above method, quantitation using a biosensor as shown above is largely affected by the oxygen concentration in the sample. Moreover, absence of oxygen in a sample solution disables the measurement.
Therefore, there is a proposed new type of glucose sensor that uses an organic compound or a metal complex, such as potassium ferricyanide, ferrocene derivatives, quinone derivatives or the like, as the electron acceptor in place of conventional oxygen. Measurement method using such new sensor oxidizes reduced form electron acceptors resulting from enzyme reaction on one electrode of the sensor in order to determine the substrate concentration, such as glucose, in the sample based on the resultant oxidation current. The use of such organic compound or metal complex as the electron acceptor enables to form a reaction layer including a known amount of GOD and electron acceptor while securing their precise and stable existence on the electrodes. Further, it enables to integrate the resultant reaction layer with the electrodes while securing an almost dry state of the reaction layer. Disposable glucose sensors based on the above technology have been drawing much attention currently. One representative is a biosensor disclosed in the Japanese Patent No. 2517153. This disposable glucose sensor facilitates measurement of the glucose concentration in a sample solution by simple insertion of the sample solution into a sensor detachably connected to a measurement device. Such measurement method is not limited to glucose quantitation and may be applicable to quantitation of other substrates in a sample.
Such prior biosensor as shown above can measure a substrate from a sample of several .mu.l. However, there is a serious demand in various fields of art recently for the development of an analysis element that facilitates measurement of a substrate from a trace amount of sample, i.e., less than 1 .mu.l. No technology, however, has so far been successful in decreasing the amount of sample drastically. Conventional measurement of, for example, sugar level has been as follows: Primarily, blood is leaked on the skin surface by puncture using a puncture-specific instrument, such as lancet, and then the blood sample-is introduced into a sensor mounted on a measurement-specific device for quantitation of blood sugar level. The use of a trace amount of sample can worsen measurement accuracy because the sample is dried before measurement. For a maximal decrease of the effect of drying on the measurement, the interval between blood sampling and sample introduction into the sensor must be shortened. The above method, however, has a limitation in shortening the interval because it necessitates individual sampling-specific and measurement-specific instruments as noted above.
Moreover, in normal conventional biosensors, a pair of electrodes are arranged on the same plane. Such structure is prone to produce inhomogeneous distribution in density of current flowing in an electrode upon voltage application onto the electrode, which can result in significant errors in measurement.